Infections with human herpesviruses are endemic and are associated with a diverse set of diseases ranging in severity from mild cold sores to life-threatening illnesses in immunocompromised patients. While some herpesvirus infections can be treated with acyclovir and similar nucleoside analogues substantial unmet medical needs remain for most herpesvirus diseases. The long-term goal of this project is to understand the mechanism of herpesvirus capsid assembly and DNA packaging. Assembly of herpesvirus capsids involves highly specific interactions of at least five different proteins and seven additional proteins are involved in DNA packaging and cleavage. The rationale for the proposed project is that due to the conserved nature of the proteins in this pathway discovery of novel antivirals that inhibit steps in cleavage/packaging may lead to a broad spectrum herpes antiviral compound. This proposal aims to extend our studies of the role of the UL25 protein in DNA packaging with regards to its functions in retention of viral DNA by binding to capsid vertices through its interaction with the UL17 protein (Aim 1). Genetic and biochemical approaches will be used to determine the role of UL28 in the assembly of a functional terminase complex and its interactions with UL15 and UL33 (Aim 2). Studies are proposed to examine the role of UL33, the smallest terminase protein, in DNA packaging and capsid nuclear egress (Aim 3). These studies utilize genetic, biochemical and structural (cryoEM) approaches to understand how the protein complexes assemble and carry out the cleavage/packaging reaction.